The yeast ribosoma1 protein L30 system is an excellent one in which to study macromolecular interactions. First, this interaction is biologically important, as the ribosomal protein regulates its own production as well as playing an essential role in the ribosome. As a repressor, it binds to a stem-internal loop-stem structure and inhibits splicing and translation. The RNA and protein determinants of binding affinity and specificity have been the focus of much study to date. The recent publication of NMR structures for the L30 complex and its components allows us to ask detailed questions about the thermodynamic contributions of various RNA protein interactions at the complex interface. In addition, there is biochemical and structural evidence for a mutually induced fit mechanism of binding. The following observations from recent work provide the starting point for this proposal. Although the RNA-protein interface is extensive, thus far alanine-scanning mutageneis only identifies three crucial resides, Phe, Lys, and Asn. To determine the nature of the disrupted contact and its thermodynamic contribution, a series of semi-conservative mutations will be made and affinities measured. Protein mutants which restore binding to mutant RNAs will be identified using a two plasmid reporter system. One critical residue involves an aromatic RNA-protein stack. If Trp can replace Phe, a fluorescence-based binding assay will be developed. This equilibrium assay may reduce reliance on efficient, but non-equilibrium gel and filter binding methods. The bound RNA is bent. Transient electric birefnngence experiments will be done to measure the RNA bend angle in the presence and absence of protein. Circular dichroism experiments may be useful in detecting conformational changes on binding, which in turn may distinguish specific from non-specific binding.